Leptospirosis is considered to be the most widespread zoonotic disease in the world. New vaccine strategies are needed for prevention of infection by these pathogenic spirochetes belonging to the genus Leptospira. The focus of this proposal is to elucidate the structural requirements and mechanisms of leptospiral outer membrane protein (OMP)-mediated immunoprotection. Leptospiral OMPs are important targets of a protective host immune response. When expressed as recombinant membrane proteins, the combination of the leptospiral porin OmpL1, and a surface-exposed lipoprotein, LipL4l are synergistically immunoprotective in the hamster model of leptospirosis. Interestingly, detergent-solubilized His6 fusion forms of these same two proteins are not immunoprotective. The goal of Specific Aim 1 is to determine what aspects of the membrane formulation are essential for the immunoprotective effect. The effects of detergent, adjuvant, protein-structure, and membrane-formulation will be systematically examined. Passive immunization studies will be performed to show that humoral mechanisms are sufficient for protection. Specific Aim 2 will establish in vitro assays of antibody-mediated protection including bactericidal activity, growth inhibition, and opsonophagocytosis. The finding of synergistic immunoprotection will be studied in Specific Aim 3 by testing for OmpL1/LipL4l interactions on the leptospiral surface, and by examining whether OmpLi/LipL4l interactions affect accessibility to antibody binding. In Specific Aim 4, polyclonal and monoclonal antibodies specific for OmpL1 and LipL4l will be tested for relevant biological activity with the goal of identifying the epitopes responsible for OMP-mediated immunoprotection.